Success Of Chimeric Antigen Receptor Research Articles

Efficient Gene Transduction and Reprogramming of Hematopoietic Cells Including T-Cells By Using a Non-Integrating Measles Virus Vector

By the ectopic expression of reprogramming genes OCT, KLF4, SOX2 and MYC (OKSM), somatic cells can be reprogrammed to induced pluripotent stem cells (iPSCs). Human iPSCs are considered a promising cell source to provide an import tool for the basic investigation and the advanced medicine including gene therapy and regenerative medicine. To establish iPSCs, integration-free Sendai virus (SV) vectors have been most widely used do far, but transduction and reprogramming of T cells without stimulation is still very challenging. On the other hand, a great success of chimeric antigen receptor T cell (CAR-T) therapies highlighted the importance of anti-cancer immunity for the cancer treatment. Particularly, many refractory patients with acute lymphoblastic leukemia and B-cell lymphoma were successfully treated with CD19-CAR-T therapies, however, some patients died before receiving the treatment due to long preparation time of CAR-Ts. Therefore, rapid production systems of CAR-Ts are desired, and for this purpose, efficient and safe gene transduction systems to T cells should be developed.In this study, we developed a new non-integrating measles virus (MV) vector-based delivery system with F deletion to eliminate cell membrane fusion-associated cytotoxicity. MV vectors transduced genes through MV receptors including CD46 and signaling lymphocyte activation molecule (CD150/SLAM). First, we examined transduction efficiencies of MV vectors and SV vectors in hematopoietic cells by using GFP expression vectors (MV-Gs and SV-Gs). Compared to SV-Gs, our MV-Gs allowed more efficient gene transfer into most hematopoietic cell type including T (3-fold) and B cells (7-fold) (Fig. 1). Furthermore, at the same multiplicity of infection (MOI) of viral transduction, MV-Gs induced less apoptosis in T cell subset compared to SV-Gs (Fig. 2) due to the slower kinetics of viral RNA amplification in the transduced cells 24 h ,48 h and 72 h post transduction.Those results encouraged us to examined if MV vectors are more potent than SV vectors in iPSC generation from unstimulated T cells. To address this question, we developed MV vectors harboring four reprogramming genes (MV-OKSMGs) and compared with SV vectors harboring these genes (SV-OKSMGs). As expected, with the MV-OKSMGs, we could generate high-quality iPSCs with the similar morphology, pluripotency markers, karyotype and differentiation capacity as human embryonic stem cells. Upon the less cytotoxicity, iPSC generation efficiency of MV-OKSMGs was much higher than that of SV-OKSMGs for unstimulated T cells (0.47 ± 0.25% vs 0.008 ± 0.009%).Considering the safe history of MV vaccine, carrying capabilities of multiple genes, more flexible receptors and higher transduction efficiency for resting T cells, our exclusive MV vector would be a potential gene transfer system for iPSC generation and lymphocyte-based-immunotherapies such as CAR-T therapies. [Display omitted] DisclosuresLiao:neopharma Japan Co. Ltd: Research Funding; TAKARA BIO, INC.: Research Funding; Shinnihonseiyaku Co., Ltd: Research Funding. Soda:Shinnihonseiyaku Co., Ltd: Research Funding; neopharma Japan Co. Ltd: Research Funding; TAKARA BIO, INC.: Research Funding. Sugawara:neopharma Japan Co. Ltd: Research Funding; Shinnihonseiyaku Co., Ltd: Research Funding; TAKARA BIO, INC.: Research Funding. Miura:neopharma Japan Co. Ltd: Research Funding; Shinnihonseiyaku Co., Ltd: Research Funding; TAKARA BIO, INC.: Research Funding. Tahara:TAKARA BIO, INC.: Research Funding. Takishima:neopharma Japan Co. Ltd: Research Funding; TAKARA BIO, INC.: Research Funding; Shinnihonseiyaku Co., Ltd: Research Funding. Hirose:TAKARA BIO, INC.: Research Funding; Shinnihonseiyaku Co., Ltd: Research Funding; neopharma Japan Co. Ltd: Research Funding. Hijikata:Shinnihonseiyaku Co., Ltd: Research Funding; neopharma Japan Co. Ltd: Research Funding; TAKARA BIO, INC.: Research Funding. Miyamoto:Shinnihonseiyaku Co., Ltd: Research Funding; TAKARA BIO, INC.: Research Funding; neopharma Japan Co. Ltd: Research Funding. Takeda:TAKARA BIO, INC.: Research Funding. Tani:neopharma Japan Co. Ltd: Research Funding; Oncolys BioPharma Inc.: Equity Ownership; SymBio Pharmaceuticals Limited: Equity Ownership; TAKARA BIO, INC.: Research Funding; Shinnihonseiyaku Co., Ltd: Research Funding.

Off-the-shelf cell therapy with induced pluripotent stem cell-derived natural killer cells

Cell therapy is emerging as a very promising therapeutic modality against cancer, spearheaded by the clinical success of chimeric antigen receptor (CAR) modified T cells for B cell malignancies. Currently, FDA-approved CAR-T cell products are based on engineering of autologous T cells harvested from the patient, typically using a central manufacturing facility for gene editing before the product can be delivered to the clinic and infused to the patients. For a broader implementation of advanced cell therapy and to reduce costs, it would be advantageous to use allogeneic "universal" cell therapy products that can be stored in cell banks and provided upon request, in a manner analogous to biopharmaceutical drug products. In this review, we outline a roadmap for development of off-the-shelf cell therapy based on natural killer (NK) cells derived from induced pluripotent stem cells (iPSCs). We discuss strategies to engineer iPSC-derived NK (iPSC-NK) cells for enhanced functional potential, persistence, and homing.

CD30-Redirected Chimeric Antigen Receptor T Cells Target CD30+ and CD30- Embryonal Carcinoma via Antigen-Dependent and Fas/FasL Interactions.

Tumor antigen heterogeneity limits success of chimeric antigen receptor (CAR) T-cell therapies. Embryonal carcinomas (EC) and mixed testicular germ cell tumors (TGCT) containing EC, which are the most aggressive TGCT subtypes, are useful for dissecting this issue as ECs express the CD30 antigen but also contain CD30-/dim cells. We found that CD30-redirected CAR T cells (CD30.CAR T cells) exhibit antitumor activity in vitro against the human EC cell lines Tera-1, Tera-2, and NCCIT and putative EC stem cells identified by Hoechst dye staining. Cytolytic activity of CD30.CAR T cells was complemented by their sustained proliferation and proinflammatory cytokine production. CD30.CAR T cells also demonstrated antitumor activity in an in vivo xenograft NOD/SCID/γcnull (NSG) mouse model of metastatic EC. We observed that CD30.CAR T cells, while targeting CD30+ EC tumor cells through the CAR (i.e., antigen-dependent targeting), also eliminated surrounding CD30- EC cells in an antigen-independent manner, via a cell-cell contact-dependent Fas/FasL interaction. In addition, ectopic Fas (CD95) expression in CD30+ Fas- EC was sufficient to improve CD30.CAR T-cell antitumor activity. Overall, these data suggest that CD30.CAR T cells might be useful as an immunotherapy for ECs. Additionally, Fas/FasL interaction between tumor cells and CAR T cells can be exploited to reduce tumor escape due to heterogeneous antigen expression or to improve CAR T-cell antitumor activity. Cancer Immunol Res; 6(10); 1274-87. ©2018 AACR.

Abstract A09: GPR64-specific CAR-transgenic T cells selectively kill Ewing sarcoma in vivo

Abstract Introduction: Pediatric cancers, including Ewing sarcoma (EwS), are only weakly immunogenic and the tumor patient’s immune system often is devoid of effector T cells for tumor elimination. Based on expression profiling technology, targetable tumor-associated antigens (TAA) are identified and exploited for engineered T-cell therapy. Here the therapeutic potential of chimeric antigen receptor (CAR) transgenic T cells directed against the G-protein coupled receptor 64 (GPR64), normally expressed only in epididymal tissue, but specifically expressed in EwS and some other sarcoma, was examined. Experimental Procedure: Two different monoclonal antibodies (mAb) directed against the extracellular region of GPR64 were generated and characterized. Subsequently, retroviral constructs containing second-generation CARs together with the scFv fragments of the respective mAbs were designed. CAR-constructs consisting of different spacers and costimulatory elements were compared (dIgG1-CD28-CD3z vs. CD8a-4-1BB-CD3z). Primary lymphocytes were transduced and tested in vitro via flow cytometry. Their activation profile and specificity was analyzed by micorarray analysis, ELISpot, and xCelligence assay as well as in immunodeficient xenograft mice. Results: Antibodies specifically stained EwS cells as determined by flow cytometry and immune histology. The signal intensity was reduced after RNAi mediated downregulation of GPR64 in EwS cell lines confirming specificity of the isolated mAbs. Further immune histology detected GPR64 expression not only in EwS but also in epithelial cancers such as renal, prostate, and pancreas carcinoma. Following sequence determination of those mAbs, two different CAR constructs were designed. Retroviruses containing such CARs transduced primary lymphocytes with high efficiency. The CAR transgenic T cells were enriched for CD8+CAR+ cells via microbead isolation and demonstrated strong proliferative capacities in vitro. Furthermore, GPR64 transmembrane cell surface target structures were specifically recognized as determined by ELISpot and xCelligence assays, but dIgG1-CD28 CAR T cells derived from one hybridoma revealed hints of early exhaustion via auto-activation. This exhaustion could be prevented by use of constructs containing CD8a-4-1BB-CD3z signaling domains for this particular hybridoma-derived scFv fragment, resulting in T-cell activation patterns more similar to TCR-transgenic T cells. Adoptive transfer of CAR-transgenic T cells into EwS tumor-bearing xenograft mice resulted in a significant suppression of tumor growth. Conclusion: CAR-transgenic T cells targeting GPR64 are a promising approach to transfer the success of CARs in hematologic malignancies to solid tumors. The cells generated in this study show strong specificity towards GPR64 and are able to control tumor cell growth. Since GPR64 expression is not restricted to EwS but also upregulated in a number of carcinomas derived from prostate, kidney, or pancreas, GPR64-specific CARs are a future treatment option for other tumor entities. Citation Format: David Schirmer, Isabel Storz, Karin Wisskirchen, Regina Feederle, Oxana Schmidt, Hinrich Abken, Ulrike Protzer, Stefan Burdach, Guenther H.S. Richter. GPR64-specific CAR-transgenic T cells selectively kill Ewing sarcoma in vivo [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr A09.

Antitumor activity of CAR-T cells targeting the intracellular oncoprotein WT1 can be enhanced by vaccination

AbstractThe recent success of chimeric antigen receptor (CAR)-T cell therapy for treatment of hematologic malignancies supports further development of treatments for both liquid and solid tumors. However, expansion of CAR-T cell therapy is limited by the availability of surface antigens specific for the tumor while sparing normal cells. There is a rich diversity of tumor antigens from intracellularly expressed proteins that current and conventional CAR-T cells are unable to target. Furthermore, adoptively transferred T cells often suffer from exhaustion and insufficient expansion, in part, because of the immunosuppressive mechanisms operating in tumor-bearing hosts. Therefore, it is necessary to develop means to further activate and expand those CAR-T cells in vivo. The Wilms tumor 1 (WT1) is an intracellular oncogenic transcription factor that is an attractive target for cancer immunotherapy because of its overexpression in a wide range of leukemias and solid tumors, and a low level of expression in normal adult tissues. In the present study, we developed CAR-T cells consisting of a single chain variable fragment (scFv) specific to the WT1235-243/HLA-A*2402 complex. The therapeutic efficacy of our CAR-T cells was demonstrated in a xenograft model, which was further enhanced by vaccination with dendritic cells (DCs) loaded with the corresponding antigen. This enhanced efficacy was mediated, at least partly, by the expansion and activation of CAR-T cells. CAR-T cells shown in the present study not only demonstrate the potential to expand the range of targets available to CAR-T cells, but also provide a proof of concept that efficacy of CAR-T cells targeting peptide/major histocompatibility complex can be boosted by vaccination.

Non-viral and viral delivery solutions for next generation cell therapy

Abstract The successes of chimeric antigen receptor (CAR) T cells in treating blood cancers have highlighted the cell therapy era. However, the difficulty of delivering molecules into immune cells has been an obstacle to more rapid advancement. Here we present an innovative large-scale Lentivirus (LV) production system as a solution to lower the cost and time of viral production. On the other hand, the next generation cell therapy will rely heavily on gene editing, especially in a safer non-viral integration manner. We have demonstrated that our novel non-viral all-in-one electroporation method provides high efficiency of gene knock-in in primary T cells. The new LV production system was developed for the clinical grade production of lentiviral vectors (LVVs) on a large-scale serum-free suspension platform. This technology employs a newly developed propriety set of GMP reagents comprising of culture media, suspension cells, transfection reagent and boosting enhancers. The system is able to deliver greater than 1 × 108 (TU/mL) functional titer with un-concentrated LVVs. For CRISPR/Cas9 gene editing in primary T cells, we were able to reach more than 90% knockout efficiency for most genes we tested, including T cell receptor (TCR), with Cas9 RNP electroporation using Neon Transfection System. More importantly, gene knock-in efficiency can be reached to greater than 30% with all-in-one electroporation, which delivers Cas9 RNP and donor DNA in one reaction using our newly developed electroporation buffer. Moreover, TCR knockout in addition to a knock-in at another locus can also be done in a single electroporation using our all-in-one method, which is ideal for developing next generation CAR and TCR-T cells.

Human CD19-Targeted Mouse T Cells Induce B Cell Aplasia and Toxicity in Human CD19 Transgenic Mice.

The clinical success of chimeric antigen receptor (CAR) Tcell therapy for CD19+ B cell malignancies can be limited by acute toxicities and immunoglobulin replacement needs due to B cell aplasia from persistent CAR Tcells. Life-threatening complications include cytokine release syndrome and neurologic adverse events, the exact etiologies of which are unclear. To elucidate the underlying toxicity mechanisms and test potentially safer CAR Tcells, we developed a mouse model in which human CD19 (hCD19)-specific mouse CAR Tcells were adoptively transferred into mice whose normal B cells express a hCD19 transgene at hemizygous levels. Compared to homozygous hCD19 transgenic mice that have ∼75% fewer circulating B cells, hemizygous mice had hCD19 frequencies and antigen density more closely simulating human B cells. Hemizygous mice given a lethal dose of hCD19 transgene-expressing lymphoma cells and treated with CAR Tcells had undetectable tumor levels. Recipients experienced B cell aplasia and antigen- and dose-dependent acute toxicities mirroring patient complications. Interleukin-6 (IL-6), interferon γ (IFN-γ), and inflammatory pathway transcripts were enriched in affected tissues. As in patients, antibody-mediated neutralization of IL-6 (and IFN-γ) blunted toxicity. Apparent behavioral abnormalities associated with decreased microglial cells point to CAR-T-cell-induced neurotoxicity. This model will prove useful in testing strategies designed to improve hCD19-specific CAR Tcell safety.

Cationic polymers for non-viral gene delivery to human T cells

The clinical success of chimeric antigen receptor (CAR) T cell immunotherapy in treating multiple blood cancers has created a need for efficient methods of ex vivo gene delivery to primary human T cells for cell engineering. Here, we synthesize and evaluate a panel of cationic polymers for gene delivery to both cultured and primary human T cells. We show that a subset of comb- and sunflower-shaped pHEMA-g-pDMAEMA polymers can mediate transfection with efficiencies up to 50% in the Jurkat human T cell line with minimal concomitant toxicity (>90% viability). We then optimize primary human T cell transfection conditions including activation time, cell density, DNA dose, culture media, and cytokine treatment. We demonstrate transfection of both CD4+ and CD8+ primary human T cells with messenger RNA and plasmid DNA at efficiencies up to 25 and 18%, respectively, with similarly high viability.

CD7-edited T cells expressing a CD7-specific CAR for the therapy of T-cell malignancies

AbstractExtending the success of chimeric antigen receptor (CAR) T cells to T-cell malignancies is problematic because most target antigens are shared between normal and malignant cells, leading to CAR T-cell fratricide. CD7 is a transmembrane protein highly expressed in acute T-cell leukemia (T-ALL) and in a subset of peripheral T-cell lymphomas. Normal expression of CD7 is largely confined to T cells and natural killer (NK) cells, reducing the risk of off-target-organ toxicity. Here, we show that the expression of a CD7-specific CAR impaired expansion of transduced T cells because of residual CD7 expression and the ensuing fratricide. We demonstrate that targeted genomic disruption of the CD7 gene prevented this fratricide and enabled expansion of CD7 CAR T cells without compromising their cytotoxic function. CD7 CAR T cells produced robust cytotoxicity against malignant T-cell lines and primary tumors and were protective in a mouse xenograft model of T-ALL. Although CD7 CAR T cells were also toxic against unedited (CD7+) T and NK lymphocytes, we show that the CD7-edited T cells themselves can respond to viral peptides and therefore could be protective against pathogens. Hence, genomic disruption of a target antigen overcomes fratricide of CAR T cells and establishes the feasibility of using CD7 CAR T cells for the targeted therapy of T-cell malignancies.

Abstract 3764: Targeting GPR64 in pediatric sarcomas with CAR transgenic T cells

Abstract Introduction: Ewing Sarcoma (ES) is the second most common bone malignancy in children and young adolescents with a high potential of dissemination into lung and bones. Patients with disseminated disease into the bone have an approximate long-term survival rate of only 10%, compelling the search for new therapeutic treatment modalities like engineered T cell therapy. Here the therapeutic potential of chimeric antigen receptor (CAR) transgenic T cells directed against G-protein coupled receptor 64 (GPR64) was examined. Experimental procedure: Two different monoclonal antibodies (mAb) directed against the extracellular region of GPR64 were generated and characterized. Subsequently, retroviral constructs containing second generation CARs together with the scFv fragments of the respective mAbs were designed. Constructs consisting of different spacers and co-stimulatory elements were compared (dIgG1-CD28-CD3z vs. CD8a-4-1BB-CD3z). Primary lymphocytes were transduced and tested in vitro via flow cytometry, ELISpot and xCelligence assay as well as in immunodeficient xenograft mice. Results: Antibodies specifically stained ES cells as determined by flow cytometry and immune histology. The signal intensity was reduced after RNAi mediated down-regulation of GPR64 in ES cell lines confirming specificity of the isolated mAbs. Following sequence determination of those mAbs two different CAR constructs were designed. Retroviruses containing such CARs transduced primary lymphocytes with high efficiency. The CAR transgenic T cells were enriched for CD8+CAR+ cells via microbead isolation and demonstrated strong proliferative capacities in vitro. Furthermore, target structures were specifically recognized as determined by ELISpot and xCelligence assays but dIgG1-CD28 CAR T cells derived from one hybridoma revealed hints of early exhaustion via auto-activation. Conclusion: CAR transgenic T cells targeting GPR64 show a promising approach to transfer the success of CARs in hematological malignancies to solid tumors. The cells generated in this study show strong specificity towards GPR64 and are able to control tumor cell growth. Since GPR64 expression is not restricted to ES but also up-regulated in a number of carcinomas derived from prostate, kidney or lung, GPR64-specific CARs may also be a future treatment option for other tumor entities. Citation Format: David Schirmer, Isabel Storz, Karin Wisskirchen, Regina Feederle, Oxana Schmidt, Hinrich Abken, Crystall Mackall, Ulrike Protzer, Stefan Burdach, Günther H. Richter. Targeting GPR64 in pediatric sarcomas with CAR transgenic T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3764. doi:10.1158/1538-7445.AM2017-3764

Unleashing the immune response against childhood solid cancers.

Tumor immunotherapy has come to the fore fuelled by impressive clinical responses to checkpoint inhibitor antibodies in a range of adult malignancies and by the success of chimeric antigen receptor T cells targeting adult and pediatric B-cell malignancies. Clearly, if appropriately fine-tuned, the immune system has the capability to seek out and destroy cancer. Studies in pediatric solid cancers so far have not shown the therapeutic potential checkpoint inhibitors described in adult cancers and this may reflect fewer tumor-associated antigens or different immune evasion mechanisms. One potential approach to overcome these limitations will be to combine interventions to undermine immune evasion mechanisms with engineered T-cell adoptive transfer.

Abstract B63: Therapeutic potential of GPR64 specific CAR T cells in pediatric sarcomas

Abstract Ewing Sarcoma (ES) is the second most common bone malignancy in children and young adolescents with a high potential of dissemination into lung and bones. Patients with localized disease receiving current treatment, have an approximate long-term survival of >65%. Patients with disseminated disease into the bone have an approximate long-term survival rate of only 10%, compelling the search for new therapeutic treatment modalities like engineered T cell therapy. Here the therapeutic potential of chimeric antigen receptor (CAR) transgenic T cells directed against G-protein coupled receptor 64 (GPR64), an orphan receptor with normal expression restricted to human epididymis and significant overexpression in ES, was examined. Therefore, two different monoclonal antibodies (mAb) directed against the extracellular region of GPR64 were generated and characterized. Subsequently, retroviral constructs containing second generation CARs together with the scFv fragments of the respective mAbs were designed. Primary lymphocytes were transduced and tested in vitro via flow cytometry, ELISpot and xCelligence assay. Antibodies specifically stained ES cells as determined by flow cytometry. The signal intensity was reduced after RNAi mediated down-regulation of GPR64 in ES cell lines confirming specificity of mAbs. Following sequence determination of those mAbs two different CAR constructs were designed. Retroviruses containing such CARs transduced primary lymphocytes with good efficiency. The CAR transgenic T cells could be enriched for CD8+CAR+ cells via microbead isolation and showed strong proliferative capacities in vitro. Furthermore, target structures were specifically recognized as determined by ELISpot and xCelligence assays. CAR transgenic T cells targeting GPR64 show a promising approach to transfer the success of CARs in hematological malignancies to solid tumors. The cells generated in this study show strong specificity towards GPR64 and are able to control tumor cell growth in vitro. Since GPR64 expression is not restricted to ES but also up-regulated in a number of carcinomas derived from prostate, kidney or lung, GPR64-specific CARs may also be a future treatment option for other tumor entities. Citation Format: Isabel Storz, David Schirmer, Karin Wisskirchen, Regina Feederle, Oxana Schmidt, Hinrich Abken, Ulrike Protzer, Stefan Burdach, Günther HS Richter. Therapeutic potential of GPR64 specific CAR T cells in pediatric sarcomas. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B63.

An oxygen sensitive self-decision making engineered CAR T-cell

A key to the success of chimeric antigen receptor (CAR) T-cell based therapies greatly rely on the capacity to identify and target antigens with expression restrained to tumor cells. Here we present a strategy to generate CAR T-cells that are only effective locally (tumor tissue), potentially also increasing the choice of targetable antigens. By fusing an oxygen sensitive subdomain of HIF1α to a CAR scaffold, we generated CAR T-cells that are responsive to a hypoxic environment, a hallmark of certain tumors. Along with the development of oxygen-sensitive CAR T-cells, this work also provides a basic framework to use a multi-chain CAR as a platform to create the next generation of smarter self-decision making CAR T-cells.

Preclinical Development of a T-Cell ALL CAR Demonstrates That Differences in CAR Membrane Distribution May Impact Efficacy

Background: Early T-cell precursor acute lymphoblastic leukemia (ETP-ALL) is an uncommon childhood leukemia that has been associated with very poor clinical outcomes in some studies. ETP-ALL cells arrest at a more immature differentiation stage than other T-lymphoblasts, and are hypothesized to retain multi-lineage differentiation potential, which may contribute to chemoresistance with standard lymphoid-directed therapy. Based on the recent clinical success of chimeric antigen receptor (CAR)-modified T-cells in children with B-ALL, we sought to identify potential surface protein targets on ETP lymphoblasts using differential gene expression analysis combined with a bioinformatic algorithm to predict surface expression.Methods: Cell-surface targets on ETP-ALL were predicted by identifying overexpressed transcripts based on gene expression and a bioinformatic algorithm to predict surface expression. Using several gene expression platforms and reference databases, (Oncogenomics website-Pediatric Oncology Branch, NCI, Gene Expression Omnibus, Gene Ontology, Human Protein References Database) ETP-ALL samples were compared to peripheral blood mononuclear cell (PBMC) controls on an individual transcript basis. A list of the top 25 transcripts was generated based on cell surface proteins, and the resultant list ordered by the degree of difference from PBMC controls. We next used human leukemia cells from six established ETP-ALL patient-derived xenograft (PDX) models using flow cytometry to evaluate for cell surface expression of proteins encoded by the overexpressed transcripts. Additionally, since CD7 and CD33 expression on ETP-ALL patient samples is universal with minimal normal tissue distribution, we developed two new second-generation anti-CD7 or anti-CD33 CAR constructs using a 41-BB/CD3ζ backbone.Results: Multiple gene transcripts encoding cell surface proteins potentially amenable to CAR T-cell targeting were overexpressed in ETP-ALL cells in comparison to PBMC controls. Many of these proteins are involved in cell signaling, cell adhesion, and metastasis, and thus potentially important for leukemic cell survival. TSPAN7 (also known as TALLA-1) was the strongest differentially expressed transcript. Despite identification of several transcripts, we did not detect increased surface expression of multiple antigens that were identified as top 25 transcripts, including TALLA-1, MCAM, EPHB6, or TSLPR. Interestingly, TALLA-1 was expressed on the more mature T-cell ALL lines, JURKAT and HPB-AU, suggesting that the surface expression of TALLA protein may be developmentally regulated. Although a new target could not be identified, given the universal expression of CD7 and CD33 on ETP-ALL, we proceeded with development of CARs targeting these antigens. CD33 CAR T-cells had excellent in vitro activity in human AML cell line MOLM-14 with minimal anti-leukemia activity in six tested ETP-ALL PDX models, perhaps due to their lower CD33 expression. We next tested T-cells transduced with a bicistronic CD7-redirected CAR with a truncated EGFR (EGFRt) to facilitate measurement of transduction efficiency and to provide a CAR deletion method. Despite high EGFRt surface expression in transduced T-cells, these CD7 CAR T-cells did not demonstrate in vitro activity against ETP-ALL or mature T-ALL samples despite high CD7 surface expression on all leukemia cell lines. We postulated that abnormal CAR distribution within the T-cell itself could be a potential factor in the observed lack of CD7 CAR T-cell activity. Using fluorescent-labeling to assess CAR surface membrane distribution, we detected high intracellular expression of the CD7 CAR, and noted that it did not traffic to the cell surface.Conclusions: We applied multimodal techniques to evaluate for cell surface expression on ETP-ALL that could serve as a target for immunotherapy. Although novel targets could not be identified, we were able to design an active anti-CD33 CAR. Further studies are in progress to evaluate what degree of antigen expression is needed to be amenable to targeted therapy. Additionally, ongoing studies are assessing whether optimization of CAR design can enhance cell surface trafficking and thereby potentially improve the anti-leukemia efficacy of CD7 CAR T-cells. DisclosuresOrentas:Lentigen Technology, Inc.: Employment. Maude:Novartis: Consultancy. Teachey:Novartis: Research Funding.

Driving an improved CAR for cancer immunotherapy

The recent clinical success of chimeric antigen receptor (CAR) T cell therapy for B cell malignancies represents a paradigm shift in cancer immunotherapy. Unfortunately, application of CAR T cell-mediated therapy for solid tumors has so far been disappointing, and the reasons for this poor response in solid tumors remain unknown. In this issue of the JCI, Cherkassky and colleagues report on their use of a murine model of human pleural mesothelioma to explore potential factors that limit CAR T cell efficacy. Their studies have uncovered the importance of the tumor microenvironment in the inhibition of CAR T cell functions, revealed a critical role for the programmed death-1 (PD-1) pathway in CAR T cell exhaustion within the tumor microenvironment, and demonstrated improved antitumor effects with a CAR T cell-intrinsic PD-1 blockade strategy using a dominant negative form of PD-1. Together, the results of this study lay the groundwork for further evaluation of mechanisms underlying CAR T cell immune evasion within the tumor microenvironment for the improvement of CAR T cell-mediated therapy for solid tumors.

393. Engineering Armored T Cell Receptor-Mimic (TCRm) Chimeric Antigen Receptor (CAR) T Cells Specific for the Intracellular Protein Wilms Tumor 1 (WT1) for Treatment of Hematologic and Solid Malignancies

Adoptive therapy with chimeric antigen receptor (CAR) T cells specific for CD19 is clinically successful in a limited set of leukemias and most CARs studied are targeted against external antigens. Wilms Tumor Antigen 1 (WT1) protein (WT1) is an intracellular antigen overexpressed in many cancers, including leukemias and solid malignancies and is thus an appealing, broadly applicable target. We have engineered the first armored T cell receptor-mimic (TCRm) CAR against WT1. Derived from the ESK1 antibody, the second generation CAR, WT1-28z, is reactive with the RMFPNAPYL peptide of WT1 that is processed and presented on the surface of cells in the context of HLA-A*02:01. WT1-28z was further modified to secrete human IL-12 cytokine, thus creating the armored CAR WT1-28z/IL-12. T cells expressing WT1-28z or WT1-28z/IL-12 are cytotoxic against a range of both hematological and solid tumors. Importantly, both WT1-directed T cells are specific for the WT1-HLA-A*02:01 complex and are not reactive against cells that do not express both HLA-A*02:01 and WT1. In established SCID/Beige mouse models of either acute leukemia or ovarian cancer, one dose of WT1-28z T cells prolongs survival of mice over untreated or irrelevant antigen-specific CAR T cell treated mice. Furthermore, one dose of the armored WT1-28z/IL12 CAR T cells further significantly prolongs survival of mice in both models over WT1-28z CAR T cell treated mice, with a subset of mice whose disease was eradicated. The armored TCRm CAR T cells against WT1 are effective in eradicating disease in both hematologic and solid tumors and may hold great clinical potential to expand on the success of CAR T cell therapy.

Generation of alloantigen-specific T regulatory cells via a chimeric antigen receptor targeting HLA-A2

Abstract CD4+ T regulatory cells (Tregs) are of clinical interest due to their potent immunosuppressive activity in both transplantation and autoimmunity models. Clinical efficacy in humans has been limited by aspects of Treg biology, including the difficulty of isolating sufficient numbers for potency without sacrificing purity and thus function. Enriching for antigen-specific Tregs greatly increases potency, however, antigen-specific Tregs are extremely rare. The success of chimeric antigen receptor (CAR) technology in generating antigen-specific effector T cells suggested that a similar approach could be used to generate alloantigen-specific Tregs for use in transplantation. We generated a new CAR specific for HLA-A2 (A2-CAR) and tested its application in the generation of alloantigen-specific human Tregs. Naïve Tregs were sorted from human peripheral blood, activated, and transduced with A2-CAR lentivirus. Stimulation through the A2-CAR upregulated Treg activation markers and functional molecules, and resulted in proliferation. Importantly, in vitro studies showed that A2-CAR-expressing Tregs maintained their expected phenotype and suppressive function, before, during and after HLA-A2-mediated stimulation. In vivo, A2-CAR Tregs were superior to Tregs expressing an irrelevant CAR at preventing xenogeneic graft-versus-host disease caused by HLA-A2+ T cells. Together, these data suggest that alloantigen-specific Tregs generated by the introduction of an A2-CAR may be a potent and specific immunosuppressive treatment in transplantation.

Regional delivery of mesothelin-targeted CAR T cell therapy generates potent and long-lasting CD4-dependent tumor immunity.

Translating the recent success of chimeric antigen receptor (CAR) T cell therapy for hematological malignancies to solid tumors will necessitate overcoming several obstacles, including inefficient T cell tumor infiltration and insufficient functional persistence. Taking advantage of an orthotopic model that faithfully mimics human pleural malignancy, we evaluated two routes of administration of mesothelin-targeted T cells using the M28z CAR. We found that intrapleurally administered CAR T cells vastly outperformed systemically infused T cells, requiring 30-fold fewer M28z T cells to induce long-term complete remissions. After intrapleural T cell administration, prompt in vivo antigen-induced T cell activation allowed robust CAR T cell expansion and effector differentiation, resulting in enhanced antitumor efficacy and functional T cell persistence for 200 days. Regional T cell administration also promoted efficient elimination of extrathoracic tumor sites. This therapeutic efficacy was dependent on early CD4(+) T cell activation associated with a higher intratumoral CD4/CD8 cell ratios and CD28-dependent CD4(+) T cell-mediated cytotoxicity. In contrast, intravenously delivered CAR T cells, even when accumulated at equivalent numbers in the pleural tumor, did not achieve comparable activation, tumor eradication, or persistence. The ability of intrapleurally administered T cells to circulate and persist supports the concept of delivering optimal CAR T cell therapy through "regional distribution centers." On the basis of these results, we are opening a phase 1 clinical trial to evaluate the safety of intrapleural administration of mesothelin-targeted CAR T cells in patients with primary or secondary pleural malignancies.

A closer look at chimeric antigen receptor specificity

The success of chimeric antigen receptor (CAR)expressing T cells as therapy for refractory hematologic malignancies (1) has spurred ongoing efforts to extend their application toward other difficult-totreat tumors. High-grade gliomas, for example, are prime candidates. These tumors—often difficult to reach by means of surgery and systemic chemotherapy—can greatly benefit from therapies that can actively home to the site of disease. Several surface proteins overexpressed by high-grade gliomas have been identified, all of which, in theory, can be targeted by CAR T cells. Interleukin (IL)13Ra2, for example, has been demonstrated to be overexpressed in high-grade gliomas (2), with very little to no expression in healthy cells. However, as the study by Krebs et al. (3) reminds us, specificity cannot be taken for granted. In the August issue of Cytotherapy, the group from Baylor College of Medicine show how CAR T cells expressing IL13 muteins (that should theoretically bind preferentially to IL13Ra2) are also capable of recognizing and responding to the closely related IL13Ra1. IL13-mutein CAR T cells produced significant levels of interferon (IFN)-g in response to both IL13Ra1 and IL13Ra2 proteins (and not to IL4R proteins). Higher levels of IFN-g (P < 0.001) were seen secreted by IL13-mutein CAR T cells after culture in plates that were pre-coated with IL13Ra1 or IL13Ra2. These IL13-mutein CART cells are also able to recognize and kill cells that express either IL13Ra1 or IL13Ra2 on their surface. Tumor cells genetically modified to express only IL13Ra1 or IL13Ra2 were able to stimulate IFN-g secretion from IL13-mutein CAR T cells, but unmodified cells or green fluorescent proteinemodified cells were not able to do so (P < 0.001). More importantly,

Abstract PR14: Fibroblast activation protein as a universal target for chimeric antigen receptor T cell therapy in solid tumors.

Abstract Rationale: The recent success of chimeric antigen receptor (CAR) T cell therapy in treating leukemia has brought new enthusiasm to this strategy. While the majority of CAR research has been primarily focused on targeting tumor cells, our group is developing a new CAR construct to target Fibroblast Activation Protein (FAP), a molecule that is highly and selectively expressed on cancer-associated fibroblasts (CAFs). It is well known that these CAFs play an important role in tumor development and tumor progression. Thus, we hypothesized that the immune-mediated destruction of CAFs by adoptively transferred FAP-CAR T cells would inhibit tumor growth in pre-clinical tumor models. Experimental procedures: The 73.3 anti-mouse FAP hybridoma was sequenced to obtain the single chain Fv to insert into our existing CAR construct with the 4-1BB and CD3ζ intracellular signaling domains. Different combinations of FAP-CAR constructs were synthesized and transduced into human T cells first to screen for optimal structure-activity response. The best two FAP-CAR constructs were then selected to transduce into mouse T cells for in vitro cytotoxicity and cytokine assays, as well as in vivo studies. To evaluate the in vivo efficacy, 2 tumor cell lines, AE17 (mouse mesothelioma) and TC1 (mouse lung cancer), were inoculated subcutaneously into flanks of syngeneic C57Bl6 mice. When the flank tumors reached 150 mm3, 10 million CAR T cells were injected through tail vein and tumors were then monitored. Tumor tissues were collected at the end of the study to evaluate the effect of CAR T cells on tumor microenvironment by flow cytometry and/or by immunohistochemistry. Results: All 8 different combinations of anti-mouse FAP CAR constructs expressed on human T cells showed target-specific activities against 3T3parental (FAP-null) versus 3T3FAP cells, in terms of cytotoxicity and cytokine production. The two best FAP-CAR constructs were then transduced into mouse T cells and re-tested for cytotoxicity and cytokine production against 3T3parental and 3T3-FAP fibroblasts, and again showed target-specific killing and cytokine production. When FAP-CAR T cells were adoptively transferred into tumor bearing mice, tumor growth inhibition (~50%) was observed in both flank tumor models while control T cells did not show any efficacy. No toxicity was detected in the animal studies. We found that FAP-CAR T cell-treated tumors had significantly more apoptotic and less proliferating tumor cells, less matrix (collagen, fibronectin), less M2 macrophages and fewer blood vessels, in comparison with the untreated and control T cell-treated tumors. To assure specificity, similar in vivo studies were also carried out in FAP knockout mice. In the KO mice, all the anti-tumor activity of FAP-CAR T cells was lost. Conclusion: Here, we demonstrate the feasibility of inhibiting tumor growth by targeting tumor stroma, instead of tumor cells, with adoptively transferred CAR T cells. We detected no safety issues with these stroma-targeted CAR T cells. We also showed that the anti-tumor effects induced by our T cells were specific to FAP. We are currently evaluating different CAR constructs against human FAP with a goal of conducting a clinical trial. This abstract is also presented as Poster A5. Citation Format: Liang-Chuan S. Wang, Albert Lo, John Scholler, Carl H. June, Ellen Pure, Steven M. Albelda. Fibroblast activation protein as a universal target for chimeric antigen receptor T cell therapy in solid tumors. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr PR14.